The α-Subunit Regulates Stability of the Metal Ion at the Ligand-associated Metal Ion-binding Site in β₃ Integrins

Background: Metal ions at LIMBS and MIDAS are essential for integrin-ligand interactions.

Results: MD simulations showed strikingly different and functionally relevant conformations of LIMBS in α_Vβ₃ and α_IIbβ₃.

Conclusion: The α-subunit regulates metal ion coordination at LIMBS and hence function of β₃ integrins.

Significance: The results reveal a new mechanism of integrin regulation by the α-subunit.

Abstract

The aspartate in the prototypical integrin-binding motif Arg-Gly-Asp binds the integrin βA domain of the β-subunit through a divalent cation at the metal ion-dependent adhesion site (MIDAS). An auxiliary metal ion at a ligand-associated metal ion-binding site (LIMBS) stabilizes the metal ion at MIDAS. LIMBS contacts distinct residues in the α-subunits of the two β₃ integrins α_IIbβ₃ and α_Vβ₃, but a potential role of this interaction on stability of the metal ion at LIMBS in β₃ integrins has not been explored. Equilibrium molecular dynamics simulations of fully hydrated β₃ integrin ectodomains revealed strikingly different conformations of LIMBS in unliganded α_IIbβ₃ versus α_Vβ₃, the result of stronger interactions of LIMBS with α_V, which reduce stability of the LIMBS metal ion in α_Vβ₃. Replacing the α_IIb-LIMBS interface residue Phe¹⁹¹ in α_IIb (equivalent to Trp¹⁷⁹ in α_V) with Trp strengthened this interface and destabilized the metal ion at LIMBS in α_IIbβ₃; a Trp¹⁷⁹ to Phe mutation in α_V produced the opposite but weaker effect. Consistently, an F191/W substitution in cellular α_IIbβ₃ and a W179/F substitution in α_Vβ₃ reduced and increased, respectively, the apparent affinity of Mn²⁺ to the integrin. These findings offer an explanation for the variable occupancy of the metal ion at LIMBS in α_Vβ₃ structures in the absence of ligand and provide new insights into the mechanisms of integrin regulation.

Introduction

Integrins are αβ heterodimeric cell adhesion receptors that mediate divalent cation-dependent cell-matrix and cell-cell adhesion during morphogenesis, as well as the maintenance of tissues and organs in adult life. 18 α- and 8 β-subunits assemble into 24 integrin receptors in mammals. Integrins regulate fundamental aspects of cell behavior, including migration, adhesion, differentiation, growth, and survival, by communicating bidirectional signals between the extracellular environment and the intracellular cytoskeleton (1).

Integrins are unusual receptors as they do not engage physiologic ligand unless activated. This property allows patrolling blood leukocytes and platelets, for example, to circulate without aggregating or interacting with the vessel walls. Inappropriate activation of integrins contributes to the pathogenesis of common diseases including heart attacks, stroke, and cancer growth and metastasis. Thus understanding how these receptors are regulated is important in promoting health and treating disease (2).

The integrin heterodimer comprises a head segment that sits on top of two leg segments each spanning the plasma membrane once and ending with a short cytoplasmic tail. The ligand-binding head consists of a seven-bladed β-propeller domain from the α-subunit that associates noncovalently with a GTPase-like domain, βA, from the β-subunit (3). Contacts between the cytoplasmic tails and transmembrane segments hold the integrin in an inactive state (unable to bind physiologic ligand). Binding of talin to the β-cytoplasmic tail breaks these contacts, switching the ectodomain into the active (ligand-competent) conformation, a process called “inside-out” signaling (4). Ligand binding then triggers global conformational changes that propagate through the plasma membrane to the cytoplasmic tails, leading to “outside-in” signaling.

Integrin-ligand interactions are regulated in a complex manner by divalent cations (5–8). Although Mn²⁺ and, to a lesser extent, Mg²⁺ stimulate ligand binding, Ca²⁺ is typically inhibitory. The ligand-binding face of the βA domain is decorated by three metal ion-binding sites: a metal ion-dependent adhesion site (MIDAS),⁴ flanked on one side (facing the propeller domain of the α-subunit) by a ligand-associated metal ion-binding site (LIMBS), and on the opposite side by an Adjacent to MIDAS (ADMIDAS) (9). A ligand aspartate completes the octahedral metal coordination of an Mg²⁺ (or Mn²⁺) at MIDAS in ligand-bound integrins. Ca²⁺ but not Mg²⁺ binds preferentially at LIMBS and ADMIDAS in physiologic buffer conditions, and both sites can coordinate Mn²⁺. The metal ion at LIMBS stabilizes the one at MIDAS (9–11), thus acting as a positive regulator of ligand binding to integrins, whereas the ADMIDAS metal ion can stabilize alternate inactive and active conformations of the integrin (2).

At the ligand-binding face of βA domain, the LIMBS loop residues Arg²¹⁶, Asp²¹⁷, and Ala²¹⁸ contact residues in the α-subunit propeller domain (Fig. 1), but a potential role of the α-subunit in regulating metal ion occupancy in the βA domain has not been explored. In this study, we carried out computational and functional studies on the two β₃ integrins α_Vβ₃ and α_IIbβ₃. Our studies reveal an important role of the α-subunit in regulating metal ion stability at LIMBS in β₃ integrins. The significance of this finding is discussed.

FIGURE 1.

α-subunit residues facing LIMBS in β₃ integrins. A ribbon diagram showing the residues from α_IIb and α_V facing LIMBS loop residues Arg²¹⁶-Ala²¹⁸ is presented. The β₃ subunits of unliganded α_IIbβ₃ (green, 3fcs.pdb) and α_Vβ₃ (yellow, 4g1e.pdb) ectodomains were superposed with Chimera. The metal ion (M²⁺) at LIMBS (sphere) has the color of the respective integrin. α_IIb and α_V residues are labeled in green and black, respectively, and the LIMBS residues are labeled in red.

EXPERIMENTAL PROCEDURES

Molecular Dynamics Simulations Design

The crystal structures of the ectodomains of α_IIbβ₃ (Protein Data Bank (PDB) 3fcs) (12) and α_Vβ₃ (PDB 4g1e) (13) were downloaded from the Protein Data Bank. In α_IIbβ₃, LIMBS (also known as SymBS, synergistic metal-binding site), MIDAS, and ADMIDAS were occupied by Ca²⁺, Mg²⁺, and Ca²⁺ respectively. Because only LIMBS was metal-occupied (by Ca²⁺) in unliganded α_Vβ₃ structure, Mg²⁺ and Ca²⁺ were respectively placed at MIDAS and ADMIDAS such that their initial distances (i.e. before minimization) from all the proximal oxygen atoms were between 2.4 and 4 Å. Mutations were made to the native structures using the software Swiss-Pdb Viewer 4.1.0 (14). All non-protein atoms were removed from α_IIbβ₃ and α_Vβ₃ structures, leaving α- and β-subunits with nearly 24,000 atoms for each, including hydrogens. Proteins were solvated in water boxes of sizes 168 × 169 × 207 Å (for α_Vβ₃) and 180 × 144 × 226 Å (for α_IIbβ₃), adding ∼183,000 and 185,000 water molecules to α_Vβ₃ and α_IIbβ₃, respectively, and ionized with 150 mm KCl. To investigate the effects of Phe¹⁹¹ in α_IIb and Trp¹⁷⁹ in α_V on coordinating divalent cations at LIMBS, six distinct combinations of cation type and mutation for α_IIbβ₃ and four for α_Vβ₃ were tested. To investigate the effects of each cation arrangement, the α_IIbβ₃ and α_Vβ₃ structures were equilibrated with LIMBS-MIDAS-ADMIDAS occupancies of Ca²⁺-Mg²⁺-Ca²⁺ and Mn²⁺-Mn²⁺-Mn²⁺.

Molecular Dynamics Simulations

MD simulations of the integrin ectodomain were performed with NAMD cvs_20130828 software package (15). The CHARMM27 force field parameter (16) was used to model the protein. The TIP3P model (17) was used for water molecules. Structures were visualized with Visual Molecular Dynamics (VMD) (18) or Chimera (19). The crystal structures of the ligand-free forms of α_IIbβ₃ and of α_Vβ₃ ectodomains were used without modification, except for the manual placement of metal ions at LIMBS and ADMIDAS in α_Vβ₃ and the removal of the sugar and water molecules before solvation. All simulations were carried out at the computing facilities of the National Energy Research Scientific Computing Center (NERSC). Periodic boundary conditions were applied in all three directions. Afterward, the entire system was minimized for 20,000 steps followed by 20 or 40 ns of equilibration. A time step of 2 fs was used in all simulations. The temperature and pressure of the systems were held constant at 1 atmosphere and 310 K respectively, using the isothermal-isobaric ensemble with the Langevin piston and Hoover method, as successfully used for modeling integrins (20–22). We performed 20 or 40 ns of MD simulation for each run, and the trajectories were used for all analyses. The cutoff distance for non-bonded interactions was 1.2 nm, and the particle mesh Ewald method was used for electrostatic force calculations (15). All B-factors were set at zero. The hydrogen atom bond length was constrained using the SHAKE algorithm (23).

Structure Analysis

Two parameters, root mean square deviation (r.m.s.d.) values of the LIMBS metal ion and the energy of interaction of the metal ion with the LIMBS pocket, were used to evaluate stability of the metal ion at LIMBS. The r.m.s.d. of a single atom is a measure of its distance at each time step from the initial position of the ion. Hence, higher r.m.s.d. values represent fluctuations with larger amplitudes and less stable ion pocket bonds. Higher interaction energies reflect higher bond stability between the ion and LIMBS or between the α-subunit and the LIMBS loop comprising residues Arg²¹⁶, Asp²¹⁷, and Ala²¹⁸. To let systems equilibrate for a considerable time span before starting to take samples for r.m.s.d. measurements, r.m.s.d. values for the ion were averaged over time steps between t = 16–20 ns for all simulations (n = 40). Energies of interaction between the divalent ion and the LIMBS pocket were estimated using Langevin dynamics. As this energy of interaction did not show significant fluctuations throughout the simulations, values for energy were averaged over t = 0–20 ns (n = 200).

Reagents and Site-directed Mutagenesis

Restriction and modification enzymes were obtained from New England Biolabs Inc. (Beverly, MA), Invitrogen Life Technologies, or Fisher Scientific. All cell culture reagents were obtained from Invitrogen Life Technologies. The non-inhibitory monoclonal antibody (mAb) AP3 (American Type Culture Collection, ATCC) detects the β₃ subunit in all conformations. The heterodimer-specific mouse mAbs CD41P2 to α_IIbβ₃ and LM609 to α_Vβ₃ were from Millipore (Danvers, MA). The function-blocking anti-β₁ mAb P5D2 was from R&D Systems, Inc. (Minneapolis, MN). Mouse mAb AP5 detects the N-terminal sequence in the PSI domain only in high affinity/ligand-bound states. The Fab fragment of AP5 was prepared by papain digestion followed by anion exchange and size-exclusion chromatography. The allophycocyanin-labeled goat anti-mouse Fc-specific IgG antibody was from Jackson ImmunoResearch Laboratories (West Grove, PA). Recombinant α_Vβ₃-specific high affinity fibronectin 10 domain (hFN10) (24) was expressed and purified from Escherichia coli as described (25), and fibronectin-depleted human fibrinogen (FB) was obtained from Enzyme Research Laboratories (South Bend, IN,). Wild-type ligands FN10 and FB and mAbs AP5 (Fab) and AP3 (IgG) were labeled respectively with N-hydroxy succinimidyl esters of Fluor 488 (Alexa Fluor 488) or Alexa Fluor 647 (Invitrogen) according to the manufacturer's instructions. Excess dye was removed using Centri-Spin size-exclusion microcentrifuge columns (Princeton Separations, Adelphia, NJ). The final hFN10, FB, AP5, and AP3 concentrations and dye-to-protein molar ratios (F/P) were determined spectrophotometrically, giving dye:protein molar ratios of 1–5. F191/W (F/W) and F992F/A992A (FF/AA) substitutions in human α_IIb, W179/F (W/F), and F990F/A990A (FF/AA) substitutions in human α_V and β-genu deletion (Δ-genu) or D158/N (D/N) substitutions in β₃ (26) were introduced using site-directed mutagenesis with the QuikChange kit (Agilent Technologies) and confirmed by DNA sequencing.

Transfections and mAb Binding

HEK293T (ATCC) cells were transiently co-transfected with pcDNA3 plasmids encoding different combinations of wild type and mutant β₃ integrins using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. Plasmids used encoded full-length wild-type α_IIbβ₃ or α_Vβ₃, the respective integrin carrying F191/W (α_IIb^F/Wβ₃) or W179/F (α_V^W/Fβ₃) substitutions or encoding constitutively active forms of these integrins by substituting the conserved transmembrane motif F992F (in α_IIb) or F990F (in α_V) to AA (27) (yielding α_IIb^F/W+FF/AAβ₃ and α_V^W/F+FF/AAβ₃, respectively) or by deleting the β-genu (Δ-genu) sequence (E⁴⁷⁶DYRPSQ) in β₃ (26) (yielding α_IIbβ₃^Δ-genu and α_IIb^F/Wβ₃^Δ-genu). In addition, two mutations were created in constitutively active α_IIbβ₃, one known to cause loss of binding to FB (Y189/A, Y/A in α_IIb) (28) (α_IIb^Y/Aβ₃^Δ-genu), and the second by substituting the LIMBS metal-coordinating residue Asp¹⁵⁸ with asparagine (α_IIb^FF/AAβ₃^D/N). 48 h after transfection, cells were detached (10 mm EDTA/PBS) and washed twice in Hanks' balanced saline solution (HBSS) and once in HBSS containing 1 mm CaCl₂/1 mm MgCl₂ (HBSS²⁺). 6 × 10⁵ α_IIbβ₃-expressing cells in 100 μl of HBSS+0.5% BSA were stained with the Alexa Fluor 647-labeled Fab fragment of AP5 (10 μg/ml; 30 min; room temperature) followed by one wash and then fixed (1% buffered paraformaldehyde). To assess β₃ integrin expression levels, 6 × 10⁵ cells in 100 μl of HBSS+0.5% BSA were incubated with CD41-P2 (anti-α_IIbβ₃) mAb or LM609 (anti-α_Vβ₃) mAb at 10 μg/ml for 30 min at 4 °C followed by one wash and then addition of allophycocyanin-labeled anti-mouse Fc-specific IgG (10 μg/ml) for 20 min on ice. Samples were again washed once in HBSS and then fixed. Other cells were labeled with Alexa Fluor 647-labeled AP3 (anti-β₃) (at 10 μg/ml; 30 min; 4 °C), washed once in HBSS, and then fixed. 20,000 cells were analyzed for each sample using a FACSCalibur or LSRFortessa flow cytometer (BD Biosciences). Binding of CD41-P2, LM609, AP3, and AP5 to β₃ integrin-expressing HEK293T was expressed as mean fluorescence intensity, as determined using the FlowJo software (BD Biosciences). Cell binding of AP5 was normalized by dividing its mean fluorescence intensity by the mean fluorescence intensity for CD41-P2 and multiplying by 100.

Soluble Ligand Binding Assays

For ligand binding experiments, 1 mm CaCl₂/1 mm MgCl₂ or 1 mm MnCl₂ was added to 6 × 10⁵ β₃ integrin-expressing HEK293T cells in 100 μl of HBSS buffer containing 0.5% BSA and incubated in the presence of a saturating amount of Alexa Fluor 488-labeled FB (160 μg/ml) or Alexa Fluor 488-labeled wild-type FN10 (12.6 μg/ml) for 30 min at 25 °C. To block any potential interaction of FN10 with endogenous β₁ integrins in HEK293T, α_IIbβ₃-expressing HEK293T cells were preincubated with the function-blocking anti-β₁ mAb P5D2 before adding Alexa Fluor 488-labeled FN10. Saturating amounts of each ligand were derived from dose-response curves, where labeled ligand was added in increasing concentrations to HEK293T cells expressing constitutively active β₃ integrins in the presence of 1 mm MnCl₂. Integrin-ligand interactions in the presence of varying concentrations of Mn²⁺ were measured by adding increasing amounts of MnCl₂ to a mixture of β₃ integrin-expressing cells and saturating amounts of Alexa Fluor 488-labeled ligand. Treated cells were then incubated with Alexa Fluor 647-labeled AP3 (10 μg/ml; 30 min; 4 °C) followed by washing once in HBSS containing the corresponding concentration of Mn²⁺. Cells were then fixed with 1% paraformaldehyde and analyzed by flow cytometry. Binding of soluble ligand to β₃ integrin-expressing cells was normalized by dividing mean fluorescence intensity by that for Alexa Fluor 647-labeled AP3 and multiplying by 100. Mean and S.D. values from three independent experiments were calculated and compared using Student's t test. Non-linear curve fittings of the dose-response curves were performed using GraphPad Prism (GraphPad Software Inc., La Jolla, CA).

RESULTS

LIMBS Structure and Stability of the Metal Ion in β₃ Integrins

Measurements of the energy of interaction of α_V and α_IIb subunits with the α-subunit-facing LIMBS loop residues Arg²¹⁶-Asp²¹⁷-Ala²¹⁸ revealed a stronger (2–3-fold) interaction of α_V with the LIMBS loop when compared with α_IIb. This difference was first detected at 8 ns of simulations and maintained through 40 ns (Fig. 2A).

FIGURE 2.

α-subunit/LIMBS interaction energies and LIMBS conformations in β₃ integrins. A, computed energy of interaction between α_IIb and α_V subunits and LIMBS loop residues Arg²¹⁶-Ala²¹⁸. The two trajectories appear to equilibrate after around 8 ns of simulation. The transient peak seen afterward (at ∼16 ns) most likely represents a high energy local minimum state that the system occasionally takes, rather than simply a random fluctuation. B and C, snapshots of molecular dynamics simulations at t = 20 ns, showing structures of LIMBS in α_IIbβ₃ (B) and α_Vβ₃ (C), in the same orientation, with the LIMBS Ca²⁺ shown as a large sphere in each case. In α_IIbβ₃, the LIMBS metal ion contacts six primary oxygens (magnified red spheres) and two secondary oxygens (small red spheres) (B). In α_Vβ₃, the LIMBS metal ion also contacts six primary oxygens but only one secondary oxygen (C). Note that the side chain of Arg²¹⁶ is stretched out in C versus in B and that five primary oxygens in α_Vβ₃ (C) lie in one plane, in contrast to the octahedral arrangement of the primary oxygens in α_IIbβ₃ (B).

MD simulations after a few nanoseconds of equilibration showed that Ca²⁺ coordination at LIMBS in α_IIbβ₃ comprised six “primary” oxygens (i.e. the two carboxyl oxygens of Asp¹⁵⁸ and Asp²¹⁷, one carboxyl oxygen of Glu²²⁰, and the carbonyl oxygen of Pro²¹⁹) (Fig. 2B), which hold a mean distance of 2.2–2.3 Å from the encapsulated cation for the entire simulation, along with two “secondary” oxygens (i.e. the carbonyl oxygens of Asp²¹⁷ and Ala²¹⁸), whose mean distance from the cation was no more than 4.5 Å over the whole trajectory. The primary oxygens remained in close contact with the Ca²⁺ at LIMBS, forming highly stable bonds with a mean length of 2.2–2.3 Å and fluctuation amplitude below 0.5 Å. The primary coordinating oxygens formed an octahedral arrangement, with a planar surface formed by OD2 of Asp¹⁵⁸, OE1 of Glu²²⁰, OD1 of Asp²¹⁷, and the carbonyl oxygen of Pro²¹⁹, with OD1 of Asp¹⁵⁸ and OD2 of Asp²¹⁷ at the top and bottom of the plane, respectively (Fig. 2B), restricting cation fluctuations. Replacing Ca²⁺ with Mn²⁺ in LIMBS, MIDAS, and ADMIDAS of α_IIbβ₃ increased the energy of interaction of Mn²⁺ with LIMBS by 5–8% and had a small but significant effect on r.m.s.d. (Table 1).

TABLE 1.

Summary of mean r.m.s.d. and interaction energy values for tested β₃ integrins

Integrin and β3 metal ion occupancy state	αIIbβ3 (Ca2+-Mg2+-Ca2+)			αIIbβ3 (Mn2+-Mn2+-Mn2+)a			αVβ3 (Ca2+-Mg2+-Ca2+)a		αVβ3 (Mn2+-Mn2+-Mn2+)a
	Wild type	F191/W	D158/N	Wild type	F191/W	D158/N	Wild type	W179/F	Wild type	W179/F
r.m.s.d. (Å)
Mean	2.0	8.3	14.5	2.7	11.8	4.7	12.3	6.1	11.0	4.7
S.D.	0.5	1.2	1.2	1.4	1.1	1.1	0.7	0.7	0.8	1.0
p valueb		<0.001	<0.001	0.002	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
Energy of interaction between LIMBS and metal ion (kcal/mol)
Mean	859	865	620	930	913	752	775	732	779	780
S.D.	20	14	14	17	23	15	15	13	15	16
p valueb		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001

^a Integrin mutation.

^b All p values were compared to wild-type α_IIbβ₃ (Ca²⁺-Mg²⁺-Ca²⁺) structure.

In contrast, the LIMBS pocket is distorted toward a planar shape in α_Vβ₃ (Fig. 2C), the result of the stronger interaction that pulled the LIMBS loop toward α_V, changing metal ion coordination at this site. MD simulations showed that Ca²⁺ at LIMBS is coordinated by six primary oxygens (the two carboxyl oxygens of Asp¹⁵⁸ and Asp²¹⁷, the carbonyl oxygen of Pro²¹⁹, and the carboxyl oxygen of Asn²¹⁵), but only one secondary coordinating oxygen, the carbonyl oxygen from Asp²¹⁷ (Fig. 2C). The carboxyl oxygens of Asp¹⁵⁸ and Asp²¹⁷ as well as the carbonyl oxygen of Pro²¹⁹ all form one planar surface that surrounds the cation, with only the side chain oxygen of Asn²¹⁵ interacting with the cation at the bottom of the plane (Fig. 2C). These changes made Ca²⁺ at LIMBS significantly less stable in α_Vβ₃ when compared with α_IIbβ₃, as reflected by the significantly higher r.m.s.d. and lower energy of interaction of the metal with LIMBS (Table 1). Replacing Ca²⁺ with Mn²⁺ in LIMBS, MIDAS, and ADMIDAS of α_Vβ₃ had minimal effects on r.m.s.d. or on the energy of interaction of Mn²⁺ with LIMBS (Table 1).

Structural Basis for the Stronger Interaction of α_V with the LIMBS Loop

In α_Vβ₃, all three LIMBS loop residues were involved in more extensive interactions with α_V: Arg²¹⁶ side chain was engaged in strong ionic bonds with Glu¹²¹ and Glu¹²³, and its carbonyl oxygen occasionally H-bonded the side chain of Tyr¹⁷⁸ (H-bond probability 0.5%). Arg²¹⁶ also formed van der Waals contacts with Phe¹⁵⁴ and with the indole group of Trp¹⁷⁹ (Fig. 3, A and B). In addition, the main and side chains of Asp²¹⁷ formed van der Waals contacts with the indole group of Trp¹⁷⁹, and the side chain of Ala²¹⁸ contacted the carboxyl oxygen of Asp²¹⁹ in α_V. These interactions stretched the LIMBS loop toward α_V, distorting LIMBS.

FIGURE 3.

α-subunit residues interacting with the LIMBS loop. A, snapshot of MD simulations at 20 ns showing interactions of the LIMBS loop Arg²¹⁶-Ala²¹⁸ residues (shown as ball and stick) with α_V subunit residues (shown as stick). The structures in A and C are shown in the same orientation after superposing LIMBS of each. LIMBS residues are labeled red in A and C (also in Figs. 4B and 5B). In α_V, the LIMBS loop interacts with the Phe¹⁵⁴, Tyr¹⁷⁸, Trp¹⁷⁹, Asp²¹⁹, Glu¹²¹, and Glu¹²³ of α_V. B, MD simulations showing van der Waals energy of interaction between Arg²¹⁶ of the LIMBS loop with Trp¹⁷⁹ and Phe¹⁵⁴ of α_V. C, snapshot of MD simulations at 20 ns showing interactions of the LIMBS loop Arg²¹⁶-Ala²¹⁸ residues with α_IIb subunit residues (shown as stick). In α_IIb, interactions are limited to the corresponding residues Tyr¹⁶⁶, Tyr¹⁹⁰, Phe¹⁹¹, and Asp²³² and a transient interaction with Glu¹²³. D, electrostatic energy of interaction between Arg²¹⁶ of the LIMBS loop and Glu¹²³ of α_IIb. Occasional jumps to higher energy levels represent ionic bonds between Arg²¹⁶ and Glu¹²³. The energy peaks at t = 7 and 16 ns show sharp increases to the same value of about 100 kcal/mol, suggesting that the ionic bond occurs at a local energy minimum that the system continues to take, whereas the Arg²¹⁶-Glu¹²³ bond spends most of the simulation time in a lower electrostatic energy state (i.e. longer bond distance).

In contrast, interaction of α_IIb with LIMBS loop residues Arg²¹⁶-Asp²¹⁷-Ala²¹⁸ was primarily limited to Arg²¹⁶. The side chain of Arg²¹⁶ formed intermittent H-bonds with the hydroxyl group of Tyr¹⁹⁰ (H-bond probability 5.0%), and its carbonyl oxygen contacted the side chain of Tyr¹⁹⁰ (Fig. 3C). Arg²¹⁶ side chain also formed occasional ionic interactions with Glu¹²³ (Fig. 3D), but made no contacts with Phe¹⁹¹. Additionally, Asp²³² made intermittent van der Waals contacts with the side chain of Ala²¹⁸.

Effects of Modifying α_IIb and α_v on Stability of the Metal Ion at LIMBS

Of the α-subunit residues contacting the LIMBS loop residues Arg²¹⁶-Asp²¹⁷-Ala²¹⁸, the side chains of Phe¹⁹¹ in α_IIb and Trp¹⁷⁹ in α_V are superimposable (Fig. 1). We evaluated the impact of interchanging these two residues on stability of the metal ion at LIMBS in the two β₃ integrins. As an internal control, we measured the effects of destabilizing the metal ion at LIMBS through the D158/N substitution (which removes one of the main coordinating oxygens from the metal ion). MD simulations showed that implementing the D158/N mutation in α_IIbβ₃ yielded a significant increase in r.m.s.d. (∼2-fold) and a reduction in the energy of interaction (Table 1), both reflecting destabilization of the metal ion at LIMBS.

Implementing the F191/W substitution in α_IIb significantly increased the energy of interaction of the LIMBS loop with the bulkier Trp¹⁹¹ in α_IIb^F/W when compared with Phe¹⁹¹ in wild-type α_IIb (Fig. 4A). Snapshot of the structure at t = 20 ns showed that the side chain of Arg²¹⁶ of β₃ releases its interaction with Tyr¹⁹⁰ and forms van der Waals contacts with the indole group of Trp¹⁹¹ in α_IIb^F/W (compare Fig. 4B with Fig. 3C), pulling the LIMBS loop region toward the α_IIb^F/W propeller domain. With Trp¹⁹¹ and Tyr¹⁹⁰ pulling the LIMBS loop in the same direction, Ala²¹⁸ is brought closer to Asp²³² to form more contacts, increasing the energy of interaction of α_IIb^F/W with the LIMBS loop and deforming the octahedral shape of the pocket. These movements displaced the oxygens forming the LIMBS pocket from their native pattern toward a more planar configuration. Although the energy of interaction of Ca²⁺ or Mn²⁺ with the LIMBS pocket in the α_IIb^F/Wβ₃ structure did not change, r.m.s.d. increased by ∼4-fold (Table 1). Hence, it appears that the energetic component of the free energy remains unchanged upon applying F191/W, whereas the entropic component is highly changed upon deformation of the pocket as represented by a 4-fold increase in r.m.s.d.

FIGURE 4.

Effect of F191/W change in α_IIbβ₃ on interaction energies and shape of LIMBS. A, computed energy of interaction between LIMBS loop residues Arg²¹⁶-Ala²¹⁸ and Trp¹⁹¹ in α_IIb^F/W. B, snapshot at t = 20 ns of the interactions of LIMBS loop with α_IIb^F/W residues Tyr¹⁶⁶, Tyr¹⁹⁰, Trp¹⁹¹, Asp²³², and Glu¹²³. Mutating Phe¹⁹¹ to Trp in α_IIb^F/W enhanced interactions of the larger Trp¹⁹¹ side chain with the LIMBS loop, especially with Arg²¹⁶, modifying the conformation of the LIMBS pocket (see “Results”).

Implementing the W179/F substitution in α_v^W/F did not change the energy of interaction of the LIMBS loop with α_V^W/F (Fig. 5A). However, it reduced fluctuations of the Ca²⁺ or Mn²⁺ at LIMBS (r.m.s.d. reduced by ∼2-fold, Table 1), the result of removing the contacts between Trp¹⁷⁹ and the LIMBS loop (Fig. 5B). The W179/F substitution did not significantly change the interaction energy of the metal ion with LIMBS (Table 1) because it did not affect the other interactions of α_V with the LIMBS loop, particularly with Arg²¹⁶ (Fig. 5B).

FIGURE 5.

Effect of W179/F in α_Vβ₃ on interaction energies and shape of LIMBS. A, computed energy of interaction between the LIMBS loop residues Arg²¹⁶-Ala²¹⁸ and Phe¹⁷⁹ in α_V^W/F. Mutating Trp¹⁷⁹ to Phe does not change the energy of interaction with the LIMBS loop significantly. B, snapshot at t = 20 ns of interactions of the LIMBS loop with α_V subunit residues Glu¹²¹, Glu¹²³, Phe¹⁵⁴, Phe¹⁷⁹, and Asp²¹⁹. The W179/F mutation weakens interaction of LIMBS loop with α_V^W/F, reshaping the LIMBS pocket. The structure is shown in the same orientation as in Fig. 4B.

Binding of Wild-type and F191/W Mutant α_IIbβ₃ to Soluble Ligand

We next sought experimental validation of the computational studies. We expressed recombinant α_IIb^F/Wβ₃ in its resting and mutationally activated (α_IIb^F/W+FF/AAβ₃, α_IIb^F/Wβ₃^Δ-genu) states in HEK293T cells and compared its surface expression, structure, and ligand binding capacity with that of constitutively active wild type α_IIb^FF/AAβ₃. As shown in Fig. 6A, surface expression of the mutant resting or constitutively active heterodimeric receptor was comparable with that of resting or constitutively active wild-type α_IIbβ₃, and the F191/W mutation did not change the recognition of the constitutively active integrin by the ligand-induced binding site (LIBS) mAb AP5 (Fig. 6B). Binding of WT α_IIbβ₃ to soluble Alexa Fluor 488-labeled FB (Alexa Fluor 488-FB) was minimal in the presence of 1 mm each of the physiologic divalent cations Ca²⁺ and Mg²⁺ (Ca²⁺/Mg²⁺), but increased only slightly in 1 mm Mn²⁺ (Fig. 6C). α_IIb^F/W+FF/AAβ₃, α_IIb^F/Wβ₃^Δ-genu bound constitutively to Alexa Fluor 488-FB in Ca²⁺/Mg²⁺-containing buffer, as expected, with 1 mm Mn²⁺ further increasing ligand binding by ∼1.5-fold (Fig. 6C).

FIGURE 6.

Effect of α_IIb F191/W mutation in α_IIbβ₃ on cell surface expression, activation, and binding to soluble ligand. A, histograms (mean ± S.D.; n = 3) comparing cell surface expression and heterodimer formation of α_IIbβ₃, α_IIb^F/Wβ₃, and constitutively active α_IIb^FF/AAβ₃, α_IIbβ₃^Δ-genu, α_IIb^F/W+FF/AAβ₃, and α_IIb^F/Wβ₃^Δ-genu. Constitutive activation reduced expression of the wild type and F191/W integrin to equivalent degrees. B, histograms (mean ± S.D.; n = 3) showing binding of the LIBS mAb AP5 to α_IIbβ₃ and to constitutively active α_IIb^FF/AAβ₃, α_IIb^F/W+FF/AAβ₃, α_IIbβ₃^Δ-genu, and α_IIb ^F/Wβ₃^Δ-genu. Binding was expressed as a percentage of binding of the heterodimer-specific mAb CD41-P2. C, histograms (mean ± S.D.; n = 3) showing binding of wild-type and constitutively active α_IIb^F/W+FF/AAβ₃ to saturating amounts of soluble Alexa Fluor 488-FB (Alex488-FB) in 1 mm Ca²⁺ plus 1 mm Mg²⁺ (Ca²⁺/Mg²⁺) or 1 mm Mn²⁺. Binding is expressed as a percentage of Alexa Fluor 647-AP3 mAb staining. F191/W did not significantly impair ligand binding to constitutively active α_IIbβ₃ in Mn²⁺. However, ligand binding to constitutively active α_IIbβ₃ in Mn²⁺ was abolished when the ligand contact residue Tyr¹⁸⁹ was simultaneously mutated to Ala.

Cellular α_IIb^F/Wβ₃ showed minimal binding to soluble Alexa Fluor 488-FB in Ca²⁺/Mg² buffer, with 1 mm Mn²⁺ increasing binding by ∼2-fold (Fig. 6C). Introduction of the F191/W mutation in constitutively active α_IIb^F/W+FF/AAβ₃ and α_IIb^F/Wβ₃^Δ-genu integrins abolished ligand binding in the presence of Ca²⁺/Mg²⁺, but did not reduce binding in the presence of 1 mm Mn²⁺ (Fig. 6C). As a negative control, replacing the ligand contact residue Tyr¹⁸⁹ in α_IIb with alanine abolished binding of cellular α_IIb^YAβ₃^Δ-genu to soluble Alexa Fluor 488-FB in 1 mm Mn²⁺(Fig. 6C), in agreement with a published study (28).

Effects of F191/W in α_IIb and W179/F in α_V on Apparent Affinity of Mn²⁺ to the Respective Integrins

We measured the binding of constitutively active wild-type and mutant β₃ integrins to saturating amount of soluble ligand across a range of Mn²⁺ concentrations. Half-maximal binding of α_IIb^FF/AAβ₃ to Alexa Fluor 488-FB was achieved at an Mn²⁺ concentration of 5.9 μm ± 2.1 (mean ± S.D., n = 3) (Fig. 7A). Removing one of the LIMBS metal-coordinating oxygens (through the D158/N mutation) increased the Mn²⁺ concentration required for half-maximal binding of ligand to α_IIb^FF/AAβ₃^D/N by ∼6-fold to 36.7 ± 8.6 μm, an indirect measure of the reduction in apparent affinity (K_d(app)) of Mn²⁺ to α_IIb^FF/AAβ₃^D/N (Fig. 7B). The F191/W substitution in α_IIb^FF/AAβ₃ yielded an almost identical value (34.6 ± 6.2 μm) (Fig. 7B). In contrast, the W179/F mutation in α_V significantly increased the apparent K_d(app) of Mn²⁺ to the α_V^W/F+FF/AAβ₃ heterodimer (as judged by mAb LM609 binding, not shown) by ∼2-fold (mean ± S.D., 5.0 ± 1.9 μm from 10.2 ± 3.5 μm to α_V^FF/AAβ₃, p = 0.018) (Fig. 7, C and D).

FIGURE 7.

Effect of α_IIb^F/W, α_V^W/F, and β₃^D/N mutations on integrin-ligand interactions in the presence of varying concentrations of Mn²⁺. A and B, dose-response curves comparing binding of α_IIb^FF/AAβ₃ (A) and α_IIb^FF/AAβ₃^D/N and α_IIb^F/W+FF/AAβ₃ (B) with saturating amounts of soluble Alexa Fluor 488-FB (Alex488-FB) in the presence of increasing concentrations of Mn²⁺. C and D, dose-response curves comparing binding of cellular α_V^FF/AAβ₃ (C) and α_V^W/F+FF/AAβ₃ (D) with saturating amounts of soluble Alexa Fluor 488-FN10 in the presence of increasing concentrations of Mn²⁺. Binding was expressed as a percentage of Alexa Fluor 647-AP3 mAb binding.

DISCUSSION

In this study, we provide computational and functional evidence that the α-subunit plays an essential role in stability of the metal ion coordination at LIMBS in β₃ integrins. By combining these two approaches, we demonstrated the following. 1) interaction of the LIMBS loop with α_V is more extensive than with α_IIb; 2) metal ion coordination at LIMBS after 20 ns of equilibration becomes planar in α_Vβ₃; 3) changing the α_IIb-LIMBS loop interface residue Phe¹⁹¹ to Trp destabilized the metal ion at LIMBS, whereas a Trp¹⁷⁹ to Phe mutation in α_V produced opposite but weaker effects; and 4) introducing F191/W in cellular α_IIbβ₃ reduced the apparent affinity of Mn²⁺ to this integrin; the reverse was observed upon introducing the W179/F mutation in cellular α_Vβ₃.

The higher energy of interaction of α_V with the LIMBS loop residues Arg²¹⁶-Ala²¹⁸ was directly related to the more extensive contacts α_V made with this loop when compared with α_IIb. The stronger contacts increased fluctuations of the metal ion at LIMBS in α_Vβ_3, as reflected by the ∼4-fold increase in r.m.s.d., and also increased the mean distances between the coordinating oxygens and the metal ion at LIMBS, as reflected by the reduction in total energy of interaction of the metal ion with the pocket. These observations may offer an explanation for the variable occupancy of LIMBS by metal ion in crystal structures of unliganded α_Vβ₃ ectodomains, where LIMBS was metal-occupied in one (4g1e.pdb, used in this study) (13) but not in four other unliganded α_Vβ₃ ectodomain structures (9, 26, 29, 30). The lack of metal occupancy at LIMBS was attributed to unfavorable crystallization conditions (13). However, LIMBS is metal ion occupied in α_Vβ₃ under the same crystallization conditions when α_Vβ₃ is ligand-bound (9, 25). The data produced in this study suggest that variability in LIMBS occupancy by metal is the result of the different contacts the LIMBS loop makes with α_V in contrast to α_IIb. In the presence of ligand, Glu²²⁰ of the βA domain provides an extra primary oxygen, stabilizing the metal ion at LIMBS. In the absence of ligand, this stabilizing influence is lost, and the metal ion is freer to escape LIMBS. This scenario is reflected in the higher r.m.s.d. and lower energy of interaction of the metal ion with LIMBS in unliganded α_Vβ₃ when compared with α_IIbβ₃ (Table 1).

α_Vβ₃ is widely expressed in tissues including bone, where it mediates dynamic cell adhesion (31, 32). The majority of Mn²⁺ in the body is sequestered in bone (33) to levels that approach the K_d of α_Vβ₃ for Mn²⁺ (7), suggesting that Mn²⁺ plays an important role in regulating α_Vβ₃ function in this tissue. In contrast, α_IIbβ₃ is solely expressed on circulating platelets in blood containing high levels of its physiologic ligands, mainly fibrinogen, and mm concentrations of the divalent cations Ca²⁺ and Mg²⁺. Maintaining α_IIbβ₃ in a dormant inactive state in this environment is therefore essential to prevent pathologic thrombosis. The present data provide insights into how regulation can be tailored to the particular environment where an integrin is expressed. Occupancy of LIMBS, MIDAS, and ADMIDAS by metal ions in unliganded α_IIbβ₃ may explain the rapid ligand association rates to activated α_IIbβ₃ (7). This potential proactivation tendency at the ligand-binding site must be counteracted by energy barrier(s) to activation elsewhere to effectively keep α_IIbβ₃ in an inactive state on circulating resting platelets. One such barrier may exist in the integrin leg segments, between the α-subunit Calf-2 domain and the β₃ subunit EGF-like 4 (IE4) and βTD domains (34). Disruption of this interface rendered α_IIbβ₃ as susceptible to Mn²⁺-induced ligand binding as α_Vβ₃ (34). In α_Vβ₃, where this Calf-2/IE4-βTD barrier is weak or absent (34), a relatively stronger α_V-LIMBS interface may help favor the inactive α_Vβ₃ conformation, thus limiting stable occupancy of the metal ion in this integrin to conditions when ligand is also accessible.